Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D263/00—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings
  • C07D263/02—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings
  • C07D263/04—Heterocyclic compounds containing 1,3-oxazole or hydrogenated 1,3-oxazole rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D277/00—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings
  • C07D277/02—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings
  • C07D277/04—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members
  • C07D277/06—Heterocyclic compounds containing 1,3-thiazole or hydrogenated 1,3-thiazole rings not condensed with other rings having no double bonds between ring members or between ring members and non-ring members with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D417/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00
  • C07D417/02—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings
  • C07D417/12—Heterocyclic compounds containing two or more hetero rings, at least one ring having nitrogen and sulfur atoms as the only ring hetero atoms, not provided for by group C07D415/00 containing two hetero rings linked by a chain containing hetero atoms as chain links
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
  • C07K5/02—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link
  • C07K5/0202—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing at least one abnormal peptide link containing the structure -NH-X-X-C(=0)-, X being an optionally substituted carbon atom or a heteroatom, e.g. beta-amino acids

Definitions

• the present invention relates to a novel tripeptide compound exhibiting an action to inhibit an enzymatic activity of a protease which originates from human immunodeficiency virus (HIV). Moreover, the present invention relates to an anti-AIDS medicine which can control in vivo growth of HIV utilizing the inhibitive activity of this novel tripeptide compound against protease originating from HIV.
• HIV human immunodeficiency virus
• HIV protease inhibitor which blocks formation and growth of infectious virus particles by inhibiting the enzymatic activity of the HIV protease can be used as an anti-virus agent.
• HIV protease inhibitors have already been reported. One of them is a transition state mimetic which is a compound something like a synthetic peptide (see, for example, T. Robins, J. Plattner, J. Acquir. Immun. Defic.
• a hydroxyethylamine derivative such as Ro31-8959 (N. A. Roberts et al., Science 248, 358-361 (1990)) which contains a phenylalanine ⁇ [CH(OH)CH 2 N] decahydroisoquinolinecarboxylic acid skeleton similar to an amino acid sequence which is selectively cleaved by an HIV protease, such as -Tyr ⁇ Pro or -Phe ⁇ Pro, and a hydroxymethylcarboxamide derivative such as a peptide derivative including a norstati skeleton such as phenylalanine ⁇ [CH(OH)C(O)N]proline (T. F. Tam et al., J. Med. Chem. 35 , 1318-1320 (1992)) are useful as an HIV protease inhibitor. Clinical application of these substances is being actively promoted.
• transition state mimetic like a transition state mimetic these compounds utilize an HIV protease inhibition activity to control production of the virus particles in host cells, whereby growth and infection of HIV can be controlled and AIDS can be prevented.
• Clinical application of these compounds as the anti-AIDS medicines is being adopted (Nakajima et al., The Pharmaceuticals monthly, Vol. 35, 2983-2989 (1993)).
• transition state mimetics are expected to become next generation anti-AIDS medicines succeeding nucleic acid derivative-type reverse transcriptase inhibitors such as AZT (azidothymidine), DDC (dideoxycytidine), and DDI (dideoxyinosine) which are already clinically used as anti-AIDS drugs.
• the present inventors have also discovered that a group of synthetic peptide compounds which are transition state mimetics including a 3-amino-2-hydroxy-4-phenylbutanoic acid residue exhibit a strong HIV protease inhibitive action and are useful as anti-AIDS medicines.
• the inventors have filed a patent application relating to the HIV protease inhibitor (Japanese Patent Application Laid-open No. 170722/1993).
• the inventors of the present invention continued the studies in which the inventors have synthesized various peptide compounds and determined chemical structures of novel compounds, including novel compounds exhibiting superior inhibition activity against HIV protease (Japanese Patent Application Laid-open No. 185631/1996, European Patent Publication EP 751145 A2).
• hydroxymethylcarboxamide derivatives having superior HIV protease inhibition activity among these peptide-type compounds require a comparatively high dose to clinically exhibit a certain effect in many cases.
• anti-AIDS medicines are continuously administered for a long period of time, development of a compound possessing a high HIV protease inhibition activity which can provide a certain effect by oral administration at a small dose is desired.
• development of a compound possessing a novel structure which is capable of tightly binding with HIV protease and exhibits a high treatment effect at a lower plasma concentration is desired.
• the present invention was completed in view of this situation and has an object of providing a novel tripeptide compound showing a high HIV protease inhibition activity as compared with an HIV protease inhibitor consisting of a transition-state mimetic peptide compound which has been proposed as a conventional anti-AIDS medicine and exhibiting superior anti-HIV virus activity accompanied by this high HIV protease inhibition activity.
• Another object of the present invention is to provide an anti-AIDS medicine comprising this novel tripeptide compound as an active component and exhibiting a treatment effect with a smaller dose.
• a tripeptide compound having a skeleton similar to an amino acid sequence which is selectively cleared by an HIV protease such as -Tyr ⁇ Pro or -Phe ⁇ Pro, particularly a hydroxymethylcarbamide derivative including a norstatin skeleton such as phenylalanine ⁇ [CH(OH)C(O)N]proline, or a hydroxyethylamine derivative including phenylalanine ⁇ [CH(OH)CH 2 N] decahydroisoquinolinecarboxylic acid, exhibits a high HIV protease inhibition activity and a high action of suppressing growth of HIV viruses, particularly when a novel structure is introduced in the C-terminal amide structure.
• A is -NH-, -NR- (wherein R represents an alkyl group having 6 or less carbon atoms), -O-CH 2 -, -CH 2 -O- or a single bond; B is -CO- or -SO 2 -; D is -CO- or -CH 2 -; E represents a divalent hydrocarbon group which may form a 5 to 7 member ring together with the adjacent nitrogen atom and carbon atom or a divalent hydrocarbon group with one or more carbon atoms therein being replaced by one or more hetero atoms, wherein said ring may form a condensed ring together with another 5 to 7 member ring or may have 3 or less substituents on that ring; R 1 is a hydrogen atom, an alkyl group having 6 or less carbon atoms, or an aromatic group or heterocyclic group having 10 or less carbon atoms, wherein the said alkyl group
• an anti-AIDS medicine comprising as an effective component a novel tripeptide compound of the following formula (II), wherein A, R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , and R 8 are the same as defined for the formula (I), X is an oxygen atom or sulfur atom, R 9 and R 10 respectively represents a hydrogen atom or a linear or branched aliphatic hydrocarbon group having 1-6 carbon atoms, or a pharmaceutically acceptable salt thereof.
• an anti-AIDS medicine comprising as an effective component a novel tripeptide compound of the following formula (III), wherein R 1 is a phenyl group which is substituted by one amino group which is either unsubstituted or substituted by one or two (mono or di) alkyl groups each having 4 or less carbon atoms and R 2 represents a linear or branched alkyl group having 1-3 carbon atoms, carbamoylmethyl group, or methylthiomethyl group; or a pharmaceutically acceptable salt thereof.
• formula (III) wherein R 1 is a phenyl group which is substituted by one amino group which is either unsubstituted or substituted by one or two (mono or di) alkyl groups each having 4 or less carbon atoms and R 2 represents a linear or branched alkyl group having 1-3 carbon atoms, carbamoylmethyl group, or methylthiomethyl group; or a pharmaceutically acceptable salt thereof.
• the tripeptide compound of the present invention is a hydroxymethylcarboxamide derivative containing a norstatin skeleton such as phenylalanine ⁇ [CH(OH)C(O)N]proline or a hydroxyethylamine derivative containing a phenylalanine ⁇ [CH(OH)CH 2 N] decahydroisoquinolinecarboxylic acid skeleton, as a transition state mimetic structure which is essential for an HIV protease inhibition activity.
• a norstatin skeleton such as phenylalanine ⁇ [CH(OH)C(O)N]proline or a hydroxyethylamine derivative containing a phenylalanine ⁇ [CH(OH)CH 2 N] decahydroisoquinolinecarboxylic acid skeleton
• this compound has a cyclic ⁇ -amino acid which contains an amino nitrogen atom in the ring as a C-terminal amino acid in the hydroxymethylcarboxamide [CH(OH)C(O)N] moiety or a hydroxyethylamine ⁇ [CH(OH)CH 2 N] moiety which constitutes a transition state mimetic structure.
• the compound also has a tripeptide structure including an ⁇ -amino acid which is substitued by an acyl group as a protective modification group on the amino group on the N-terminal side of the dipeptide structure which constitutes the transition state mimetic structure.
• the cyclic ⁇ -amino acid at the C-terminal is an ⁇ -aminocarboxamide and the nitrogen atom of carbamoyl group of this ⁇ -aminocarboxamide has a substituted or unsubstituted benzyl group.
• the compounds shown by the general formula (III), on the other hand, is characterized by the ⁇ -amino acid group at the N-terminal, as well as by the selection of the acyl group which is used as a protective modification group on that amino group.
• hydroxymethylcarboxamide [CH(OH)C(O)N] moiety and the hydroxyethylamine [CH(OH)CH 2 N] moiety which constitute the transition state mimetic structure in the tripeptide compound shown by the general formula (I) of the present invention are respectively present as a 3-amino-2-hydroxy-4-substitued butanoyl skeleton [-CH 2 CH(NH)CH(OH)C(O)-N] or a 3-amino-2-hydroxy-4-substituted butyl skeleton [-CH 2 CH(NH)CH(OH)CH 2 -N].
• the steric configuration of (2S,3S) isomer is preferred for the 3-amino-2-hydroxy-4-substitued butanoyl moiety [-CH 2 CH(NH)CH(OH)C(O)-N], whereas the steric configuration of (2R, 3S) isomer is preferred for the 3-amino-2-hydroxy-4-substituted butyl moiety [-CH 2 CH(NH)CH(OH)CH 2 -N].
• a preferred steric configuration is (L)-isomer for the cyclic ⁇ -amino acid in which the divalent group E constitutes the ring-forming group.
• (L)-isomer is also preferred for the ⁇ -amino acid having R 2 group as a side chain which forms a peptide bond at the 3 position amino group of said substituted butanoyl moiety or substituted butyl moiety.
• the group which is conventionally selected for the transition state mimetic structure in a peptide compound which possesses this kind of HIV protease inhibition activity can be utilized as the group R 3 which is replaced on the 4 position of 3-amino-2-hydroxy-4-substitued butanoyl skeleton or 3-amino-2-hydroxy-4-substitued butyl skeleton.
• the group R 3 is selected from the groups which possess a substituent on an aryl group, aryloxy group or arylthio group, or on an aromatic ring included in these groups, among the groups in which the cyclic group wherein such a bonding radical is present is a 6 member cyclic group.
• Phenyl group, naphthyl group, phenylthio group, phenoxy group, and the like can be given as examples of R 3 .
• a monocyclic or dicyclic group, particularly a monocyclic group, is preferred as the aromatic ring which constitutes this aryl group and the like.
• the number of substituent which is present on the aromatic ring of the group R 3 is preferably two or less, and more preferably one.
• An alkyl group having 4 or less carbon atoms, alkyloxy group, alkylamino group, halogeno group, hydroxyl group, amino group, and the like can be given as examples of the substituent.
• the halogeno group includes chloro group, bromo group, iodine group, and fluoro group, with particularly preferred being chloro or and fluoro group.
• a phenyl group, phenoxy group, phenylthio group, and the like are given as preferred examples of the group R 3 , with the phenyl group being particularly preferred.
• this group E is a divalent hydrocarbon which may form a 5 to 7 member ring together with the adjacent nitrogen atom and carbon atom or a divalent hydrocarbon group with one or more carbon atoms in the 3-5 carbon chain of said hydrocarbon group being replaced by one or more hetero atoms, wherein said ring may form a condensed ring together with another 5 to 7 member ring or may have 3 or less substituents on that ring.
• the hetero atom which may replace for the carbon atom in the divalent group E is a nitrogen atom, sulfur atom, or oxygen atom, wherein the sulfur atom may be present as a thio group, sulfinyl group, or sulfonyl group.
• the ⁇ -amino group which is formed by the group E it is desirable that this hetero atom is bonded to neither the nitrogen atom which forms the amino group at the ⁇ -position nor the carbon atom at the ⁇ -position.
• the cyclic group which is formed by the group E together with the adjacent nitrogen atom and carbon atom may form a cyclic structure by condensation with another 5-7 member ring.
• the group E may be either a single ring or may form a two or more rings, when the group E is a group consisting of two or more rings formed by condensation, ortho-condensation is desirable.
• this cyclic group may possess three or less other substituents on the linear chain of this group E.
• the groups to be substituted here include a linear or branched aliphatic hydrocarbon group having 1-6 carbon atoms, aromatic hydrocarbon group, heteroaromatic group, hydroxyl group, a linear or branched aliphatic hydrocarbonoxy group having 1-6 carbon atoms, and halogeno group.
• the total number of the substituent is preferably two or less.
• both of these two substituents may be members of a cyclic structure.
• the two substituents may take a cross-linking structure or may be present as a dicyclic group in which the two rings combine by a spiro bond.
• the substituents may form a double bond such as an oxo group.
• proline and derivatives of proline having a substituent on the 4 position thereof such as 4-hydroxyproline, 4-benzyloxyproline, 4-phenylproline, 4-benzylproline, 4-methylthioproline, 4-phenylthioproline, 4-fluoroproline, 4-chloroproline, and the like can be given.
• the compounds which condense by cyclization with other 5-7 member cyclic groups are the compounds which cyclize with a cycloalkane, such as octahydroindole-2-carboxylic acid, octahydroiso-indole-1-carboxylic acid, 2-azabicyclo[3.3.0] octane-3-carboxylic acid; the compounds which cyclize with an aromatic group or hetero-aromatic group, such as indoline-2-carboxylic acid, iso-indoline-1-carboxylic acid, and the like.
• a cycloalkane such as octahydroindole-2-carboxylic acid, octahydroiso-indole-1-carboxylic acid, 2-azabicyclo[3.3.0] octane-3-carboxylic acid
• an aromatic group or hetero-aromatic group such as indoline-2-carboxylic
• ⁇ -amino acid corresponding to the 5 member cyclic group which selects a divalent group produced by substitution of one carbon atom contained in a linear hydrocarbon group having three carbon atoms with a hetero atom as the group E, 1,3-oxazolidine-4-carboxylic acid and 1,3-thiazolidine-4-carboxylic acid, as well as 5-methyl-1,3-oxazolidine-4-carboxylic acid, 5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, and the like which possess a substituent on this 5 member heterocyclic group, can be given.
• ⁇ -amino acid corresponding to the 6 member cyclic group which selects a linear hydrocarbon group having four carbon atoms for the group E pipecolic acid (2-piperidinecarboxylic acid) can be given.
• the compounds which condense by cylization with other 5-7 member cyclic groups are decahydroisoquinoline-3-carboxylic acid and decahydroisoquinoline-1-carboxylic acid which cyclize with a cycloalkane, as well as 1,2,3,4-tetrahydro isoquinoline-3-carboxylic acid, 1,2,3,4-tetrahydro isoquinoline-1-carboxylic acid, and the like which cyclize with an aromatic group or hetero-aromatic group.
• Piperazine-2-carboxylic acid and the like can be given as examples of the ⁇ -amino acid corresponding to the 6 member cyclic group which selects a divalent group produced by substitution of one carbon atom contained in a linear hydrocarbon group having four carbon atoms with a hetero atom as the group E.
• cyclic groups 5 or 6 member monocyclic groups, particularly 5 member monocyclic groups, are preferable in the hydroxymethylcarboxamide [CH(OH)C(O)N] type compound.
• the compound shown by the following general formula (XI) is preferable. wherein X is an oxygen atom or sulfur atom, and R 9 and R 10 are respectively a hydrogen atom or aliphatic hydrocarbon group having 1-6 carbon atoms.
• 1,3-oxazolidine-4-carboxylic acid and 1,3-thiazolidine-4-carboxylic acid which are the compounds having a hydrogen atom or a methyl group respectively for the groups R 9 and R 10 in the ⁇ -amino acid constituting this 5 member cyclic group, as well as 5-methyl-1,3-oxazolidine-4-carboxylic acid, 5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid, and the like.
• cyclic groups 5 or 6 member cyclic groups, particularly 6 member cyclic groups, are preferable in the hydroxyethylamine [CH(OH)CH 2 N] type compound.
• the compound shown by the following general formula (XII) is preferable.
• Y is a carbon atom or nitrogen atom
• R 11 and R 12 are respectively a hydrogen atom, aliphatic hydrocarbon group which may have a branched chain, or an aromatic hydrocarbon group which may possess a substituent or a group derived from the aromatic hydrocarbon group by substitution of a hetero atom, provided that R 11 and R 12 together may form a ring.
• pipecolic acid (2-piperidinecarboxylic acid) or piperazine-2-carboxylic acid constituting a 6 member cyclic group as well as a compound with two or less substituents which may be linear or branched aliphatic hydrocarbon groups having 1-6 carbon atoms substituting on this 6 member cyclic group or a compound condensed by cyclization with another 5-7 member cycloalkane, such as decahydroisoquinoline-3-carboxylic acid or decahydroisoquinoline-1-carboxylic acid, are preferred.
• the amino acid residue having the group R 2 as a side chain can also be used inasmuch as the same is conventionally selected as an amino acid corresponding to the P2 position of a substrate in a transition state mimetic structure in the peptide compounds possessing this type of HIV protease inhibition activity. Therefore, the amino acid having a steric configuration corresponding to the (L)-isomer is usually desirable.
• an aliphatic hydrocarbon group having 1-7 carbon atoms or an aromatic hydrocarbon group can be used for R 2 .
• This aliphatic hydrocarbon group may be either linear or branched.
• the carbon atom constituting the bone of the aliphatic hydrocarbon group or the aromatic hydrocarbon group may be replaced by a hetero atom.
• such a carbon atom may be replaced even by a carbamoyl group, carboxyl group, or halogeno group.
• the aliphatic hydrocarbon group having 1-7 carbon atoms used as R 2 includes linear or branched alkyl groups such as methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, as well as unsaturated hydrocarbon groups having the same carbon skeleton as these alkyl groups but including a carbon-carbon double bond, and hydrocarbon groups having a cyclic structure such as cyclohexylmethyl group, and the like.
• Hetero atoms which may be replaced for the skeletal carbon of these aliphatic hydrocarbon groups may be an oxygen atom, sulfur atom, nitrogen atom, and the like.
• groups replaced by an oxygen atom are those having an ether structure such as methoxymethyl group, methoxyethyl group, and the like, and those having a hydroxyl group structure such as 1-hydroxyethyl group.
• groups replaced by a sulfur atom the groups having a thioether structure such as methylthiomethyl group, methylthioethyl group, and the like are given.
• a group with a nitrogen atom at the terminal forming a cyano group is also included.
• groups replaced by a carbamoyl group as substituent such as a carbamoylmethyl group, carbamoylethyl group, or by a halogeno group such as chloro group, bromo group, iodine group, fluoro group, trifluoromethoxyethyl group, trifluoromethyl group, can be given.
• the aromatic hydrocarbon group in the group R 2 is the hydrocarbon group which possesses an aromatic ring, for example, an aromatic ring group such as phenyl group, or a compound having a bonding radical in a side chain hydrocarbon group which is present on the aromatic ring such as, for example, benzyl group.
• a nitrogen atom, oxygen atom, and sulfur atom are included in the hetero atoms which may replace for the skeletal carbon of these aromatic hydrocarbon groups.
• the groups having an imidazole structure, furan structure, or thiophene structure which forms a 5 member cyclic structure and exhibits aromaticity are included. As a matter of course, substitution by two or more hetero atoms is acceptable.
• substituents which may be present not only on the aromatic ring, but also in a hydrocarbon group of side chains include a carbamoyl group and a halogeno group such as chloro group, bromo group, iodine group, or fluoro group.
• any groups which have been deemed conventionally to be desirable according to the types of a dipeptide structure forming a transition state mimetic structure which combines with the C-terminal are preferably used as the group R 2 .
• preferred examples of the group R 2 include a methyl group, ethyl group, propyl group, isopropyl group, butyl group, isobutyl group, sec-butyl group, tert-butyl group, carbamoylmethyl group, carbamoylethyl group, methylthiomethyl group, methylthioethyl group, methoxymethyl group, methoxyethyl group, trifluoromethyl group, trifluoroethyl group, benzyl group, cyclohexylmethyl group, and the like.
• R 4 , R 5 , R 6 , R 7 , and R 8 are respectively a hydrogen atom or an alkyl group having 3 or less carbon atoms, a halogeno group, amino group, hydroxyl group, aminomethyl group, hydroxymethyl group, or a mono- or di-alkyl substitued amino group replaced by an alkyl group having 3 or less carbon atoms.
• the halogeno group may be a chloro group, bromo group, or fluoro group, and the alkyl group having 3 or less carbon atoms may have a branched chain.
• substituted benzyl groups those having a substitution group at the ortho position, specifically either in the group R 4 or R 8 , are particularly desirable.
• a small group such as a methyl group, ethyl group, halogeno group, or the like, is desirable as the group R 4 or R 8 which are replaced at the ortho position.
• 2-methylbenzyl group, 2-chlorobenzyl group, 3-amino-2-methylbenzyl group, 5-amino-2-methylbenzyl group, 3-hydroxy-2-methylbenzyl group, 2,6-dimethylbenzyl group, 2,3-dimethylbenzyl group, 2,5-dimethylbenzyl group, and the like can be given as preferred substituted benzyl groups.
• 2-methylbenzyl group and 2-chlorobenzyl group in which a methyl group or halogeno group, preferably a methyl group or chloro group, is a substitution group at the 2 position are particularly desirable as the mono-substituted benzyl group.
• the methyl group or halogeno group, particularly the methyl group or chloro group, is preferable also as a substitution group at the 2 position even in the multi-substituted benzyl group having substituents in addition to the 2 position.
• the atomic group R 1 -A-B- existing in the amino group at the N-terminal which corresponds to the P3 position in the substrate plays a role of protecting and modifying the N-terminal amino group of the tripeptide compound of the present invention.
• the bivalent group B forms a bond between the N-terminal amino group and the amide type. This group is either a carbonyl group (-CO-) or sulfonyl group (-SO 2 -).
• the bivalent group A is an imino group (-NH-), a lower alkyl group-substituted imino group (-NR-, wherein R is an alkyl group having 6 or less carbon atoms), oxymethylene group (-O-CH 2 -), or methyleneoxy group (-CH 2 -O-), or a single bond.
• the group R 1 bonding to the N-terminal amino group via the group -A-B- is a hydrogen atom, an alkyl group having 6 or less carbon atoms, aromatic group or heterocyclic group having 10 or less carbon atoms.
• the alkyl group may have an alkyloxy group having 4 or less carbon atoms as a substituent, and the aromatic group or heterocyclic group may have a substituent selected from an alkyl group having 4 or less carbon atoms, alkyloxy group, mono or dialkyl-substituted amino group, halogeno group, hydroxyl group, amino group, or alkyl or alkenyl group having 3 or less carbon atoms substituted by a monocyclic aromatic or monocyclic heteroaromatic group.
• the alkyl group having 6 or less carbon atoms in R 1 may be either linear or branched.
• the aromatic group or heterocyclic group having 10 or less carbon atoms in R 1 may be either monocyclic or dicyclic, and includes the groups having 10 or less carbon atoms which constitute this cyclic structure.
• the number of atoms constituting each ring is not limited to 6 inasmuch as the group has aromaticity.
• a heterocyclic group possessing aromaticity e.g. a group made from a 5 member ring and a 6 member ring by cyclic condensation, such as benzofuran group or benzothiophene group, is acceptable.
• the groups constituting a single conjugated system including an oxo oxygen atom of quinone such as a naphthoquinone structure, for example are also included.
• a skeleton with an atom outside the ring which forms a double bond with an atom existing on the ring and produces a skeleton with a conjugated system showing aromaticity such as, for example, a chromone moiety is also acceptable. Inasmuch as there is at least one ring exhibiting aromaticity, the addition of a hydrogen to an unsaturated bond is allowable.
• any atomic groups R 1 -A-B- which have been deemed conventionally preferable according to the types of the tripeptide moiety to which these groups combine, specifically according to the types of a dipeptide structure forming a transition state mimetic structure, are preferable also in the present invention.
• the aromatic group or heterocyclic group having 10 or less carbon atoms replaced for the group R 1 among the alkyl group having 4 or less carbon atoms, alkyloxy group, and mono- or di-alkyl-substituted amino group, the alkyl group may be either linear or branched and the halogeno group means chloro group, bromo group, or fluoro group.
• the alkyl or alkenyl group having 3 or less carbon atoms substituted by a monocyclic aromatic or monocyclic heteroaromatic group which can be present as a substitution group on the aromatic group or heterocyclic group having 10 or less carbon atoms includes an alkyl or alkenyl group having 3 or less carbon atoms substituted by a monocyclic aromatic ring group, specifically, by a phenyl group having a side chain, such as a phenyl group, tolyl group, xylyl group, or mesityl group, or an alkyl or alkenyl group having 3 or less carbon atoms substituted by a monocyclic heteroaromatic group, for example, a 5 member group such as a furyl group or 6 member group such as a pyridyl group.
• these are benzyl group, phenethyl group, cinnamyl group, or the like.
• an alkyl or alkenyl group having 3 or less carbon atoms substituted by a monocyclic heteroaromatic group is included as a substitution group, it is preferable that the aromatic or heteroaromatic ring which is a moiety of the group R 1 be a monocyclic group and the atomic group R 1 -A-B- should not be a large group. Therefore, when an alkyl or alkenyl group having 3 or less carbon atoms substituted by a monocyclic aromatic group or a monocyclic heteroaromatic group is included as a substitution group, the number of this group is preferably one.
• the atomic group R 1 -A-B- as a whole is preferably an acyl group with a chain structure, such as an alkoxylacetyl group wherein A is an oxymethylene group (-O-CH 2 -) and B is a carbonyl group (-CO-), an alkoxylcarbonyl group wherein A is a methyleneoxy group (-CH 2 -O-) and B is a carbonyl group (-CO-), an alkanoyl group wherein A is a single bond and B is a carbonyl group (-CO-), or an alkoxylalkoxylcarbonyl group which is a group with an alkyloxy group having 4 or less carbon atoms substituted on the group R 1 of these groups.
• the chain structure may be branched, but the number of the branch is preferably one.
• the group wherein B is a carbonyl group (-CO-) is more preferable than the group wherein B is a sulfonyl group (-SO 2 -).
• the tripeptide compound shown by the following general formula (II) can be given as still another preferred embodiment of the hydroxymethylcarboxamide [CH(OH)C(O)N] type compound: wherein A, R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , and R 8 are the same as defined in the above general formula (I), X denotes an oxygen atom or sulfur atom; and R 9 and R 10 respectively represent a hydrogen atom or a linear or branched aliphatic hydrocarbon group having 1-6 carbon atoms.
• the compound with a sulfur atom for X in the general formula (II) is a particularly desirable compound.
• the tripeptide compound shown by the following general formula (IV) can be given as still another preferred embodiment of the hydroxyethylamine [CH(OH)CH 2 N] type compound, wherein A, R 1 , R 2 , R 4 , R 5 , R 6 , R 7 , and R 8 are the same as defined in the general formula (I), Y is a carbon atom or nitrogen atom, and R 11 and R 12 are respectively a hydrogen atom, a linear or branched aliphatic hydrocarbon group, an aromatic hydrocarbon group which may have a substituent, or a group derived from such an aromatic group by substitution of a hetero atom, provided that R 11 and R 12 may form a bond together.
• tripeptide compounds represented by the following general formula (VI), (VII), and (VIII): wherein R 1 is the same as defined in the general formula (I), R 2 is a methyl group, ethyl group, propyl group, isopropyl group, carbamoylmethyl group, or methylthiomethyl group, and R 4 is a methyl group or a chloro group, are given as particularly preferred embodiments of the coumpounds having the above-described general formula (V).
• Particularly preferred compounds represented by the general formula (VI), (VII) or (VIII) are those having a phenyl group, 2-chromone group, benzofuran-2-yl group, 5-isoquinolyl group, benzothiazol-2-yl group, 2-quinolyl group, benzimidazol-2-yl group, or the like for the group R 1 .
• Specific examples include:
• the compound represented by the general formula (III) possesses a form similar to the compound represented by the above general formula (VII) except that a substituent on the C-terminal amide nitrogen atom is a tert-butyl group instead of a substituted benzyl group.
• R 2 is selected from a methyl group, ethyl group, propyl group, isopropyl group, carbamoylmethyl group, or methylthiomethyl group as well as R 2 in the above-mentioned general formula (VII).
• R 2 is selected from an ethyl group, propyl group, isopropyl group, carbamoylmethyl group, and methylthiomethyl group.
• One feature characterizing the compound represented by the general formula (III) is that a substituted phenoxyacetyl group exists on the N-terminal amino group of ⁇ -amino acid residue having the group R 2 as the side chain as a protective modification group.
• the substituted phenoxyacetyl group which is R 1 mono-substituted phenyl group which is substituted by an amino group or mono- or di-alkyl substituted amino group having 4 or less carbon atoms, and preferably mono-substituted phenyl group in which mono- or di-alkyl substituted amino group substitutes on the 3 position thereof is selected.
• the following compounds in which a phenyl group is selected as R 1 wherein a methylamino group or a dimethylamino group substitutes on the 3 position thereof, can be given:
• the pharmaceutically acceptable salt of the tripeptide compound of the present invention includes a substituent which substitutes on a substituted benzyl group or a cyclic group of R 1 , which exists in the compound, or, for example, in the case where a basic nitrogen atom is present in the other condensed rings, a salt which is formed from the nitrogen atom and various pharmaceutically acceptable acid, and specifically, a pharmaceutically acceptable salt such as a hydrogen chloride, acetic acid salt, or methanesulfonate.
• Pharmaceutically acceptable salts formed from a phenolic hydroxyl group which substitutes on a substituted benzyl group and the like and various pharmaceutically acceptable mono-valent cations are also given as examples.
• a typical example of such a pharmaceutically acceptable salt is a sodium salt.
• the tripeptide compounds of the present invention represented by the general formula (I) are composed of (i) a tripeptide skeleton which is indispensable for the HIV protease inhibition activity and consists of a dipeptide moiety in which the substituted benzyl group substitutes on a nitrogen atom of amide at the C-terminal and an ⁇ -amino acid residue which is present at the N-terminal, and (ii) an atomic group which protectively substitutes the N-terminal ⁇ -amino group of the tripeptide skeleton.
• an intermediate material compound represented by the following general formula (XIII), wherein D, E, R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 are the same as defined in the general formula (I) and A 2 is a protective group commonly used with an amino group, which forms the dipeptide moiety is previously prepared according to the following processes by a conventionally known method.
• the tripeptide skeleton represented by the following general formula (XIV) is obtained by an elongation of the ⁇ -amino acid residue of which R 2 is the side chain to the N-terminal amino group of the intermediate material.
• D, E, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 are the same as defined in the general formula (I) and A 3 is a protective group commonly used with an amino group.
• the atomic group R 1 -A-B is introduced into the N-terminal amino group of the tripeptide skeleton by an N-acylation and the like, whereby the intermediate material compound can be prepared.
• the commonly used protective group for the amino groups represented by A 2 and A 3 a protective group which is easily released by an acid treatment is used. Process for preparing the hydroxyethylamine derivatives and hydroxymethylcarboxamide derivatives will be described in more detail.
• This compound corresponds to an intermediate material in a synthetic method of a hydroxyethylamine type HIV protease inhibitor, of which the preparation method has reported in various publications (see, for example, Science 248 , 358-361 (1990), N. A. Roberts et al., B. M. Kim et al., Bioorg. & Med. Chem. Lett. 4 , 2273-2278 (1994)).
• a substituted benzylamine represented by the following general formula (XVI), wherein E, R 4 , R 5 , R 6 , R 7 , and R 8 are the same as defined in the general formula (I) are reacted according to a conventional method to prepare an amide bond, thereby obtaining a cyclic ⁇ -aminocarboxamide derivative with protected N-terminal which is represented by the following general formula (XVII), wherein E, R 4 , R 5 , R 6 , R 7 , and R 8 are the same as defined in the general formula (I) and A 1 is a commonly used protective group for an amino group of the general formula (XV).
• N-terminal protected aminoepoxide derivative represented by the following general formula (XVIII), wherein R 3 is the same as defined in the general formula (I) and A 2 is a commonly used protective group for an amino group, in a solvent such as iso-propanol while heating under refluxing to ring-open the epoxide with the amine, thereby obtaining an amino alcohol derivative represented by the following general formula (XIX), wherein E, R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 are the same as defined in the general formula (I) and A 2 is a commonly used protective group for an amino group of said general formula (XVIII).
• a dipeptide intermediate material represented by the following general formula (XX) in which a methylene group is selected as the group D in the general formula (XIII) can be obtained.
• E, R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 are the same as defined in the above-mentioned general formula (I).
• the steric configuration of the resulting dipeptide intermediate material is also the (2R,3S)-type.
• the atomic group represented by A 5 is derived from a reagent for synthesizing the peptide.
• This ⁇ -amino acid anhydride, active ester derivative, or the like which is represented by the general formula (XXII) is reacted with the intermediate material which is represented by the general formula (XX) in a solvent such as N,N-dimethylformamide (DMF) to obtain a tripeptide skeleton represented by the following general formula (XXIII), wherein E, R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 are the same as defined in the general formula (I) and A 3 is a commonly used protective group for an amino group of the general formula (XXI).
• the atomic group R 1 -A-B- is an acyl group type
• the atomic group R 1 -A-B- can be introduced by the following N-acylation, for example.
• a carbodiimide such as DCC (N,N'-dicyclohexylcarbodiimide)
• an acid anhydride such as an acetic anhydride or trifluoroacetic anhydride
• the acid anhydride of the carboxylic acid represented by the general formula (XXV) is reacted with the above-described intermediate material with the group A 3 removed represented by the general formula (XXIII) in which a methylene group is selected as the group D in a solvent such as N,N-dimethylformamide (DMF) to obtain the target N-acylated hydroxyethylamine type derivative represented by the general formula (IX).
• a solvent such as N,N-dimethylformamide (DMF)
• the atomic group R 1 -A-B- of which the group B is a sulfonyl group which becomes a sulfonamide by forming a bond with the N-terminal amino group can be introduced by a similar method to the above-mentioned N-acylation such as a method using a sulfonyl chloride or a method using a sulfonic acid anhydride and active ester derivative.
• the intermediate material compound corresponds to an intermediate material in a synthetic method of a hydroxymethylcarboxamide type HIV protease inhibitor and the preparation method has been reported in various publications (see, for example, Yoshiaki Kiso, Journal of Synthetic Organic Chemistry Japan vol. 52, 403-412 (1994)).
• an ⁇ -amino-carboxamide derivative represented by the general formula (XVII) is subjected to a condensation with an N-protected derivative of 3-amino-2-hydroxy-4-substituted butanoic acid represented by the following general formula (XXVI), wherein R 3 is the same as defined in the general formula (I) and A 2 is a common protective group for an amino group releasable with an acid, using a carbodiimide such as DCC or EDC and an additive compound such as HONB (N-hydroxy-norbornene-2,3-dicarboxyimide) or HOBt (N-hydroxybenzotriaole) to form a peptide bond, thereby obtaining an N-protected dipeptide derivative represented by the following general formula (XXVII), wherein E, R 3 , R 4 , R 5 , R 6 , R 7 , and R 8 are the same as defined in the general formula (I) and A 2 is the same group as the common
• an intermediate material represented by the general formula (XIII) from which A 2 has been removed and a carbonyl group is selected as the group D can be obtained.
• the (2S-3S)-derivative for example, is used for a steric configuration of the 3-amino-2-hydroxy-4-substituted butanoic acid N-protected derivative, the steric configuration of the resulting dipeptide intermediate material is also the (2S-3S)-type.
• an optical isomer having the same steric configuration as the raw material can be obtained.
• the target hydroxymethylcarboxamide type derivative represented by the general formula (X) can be obtained by reacting the intermediate material represented by the general formula (XIII) prepared in the step 2-1 in which a carbonyl group is selected as the group D, with an amino acid represented by the general formula (XXII), then with a carboxylic acid represented by the general formula (XXIV).
• the hydroxyethylamine type derivative represented by the general formula (IX) or the hydroxymethylcarboxamide type derivative represented by the general formula (X) which are prepared by the above process can be used as the HIV protease inhibitor by removing impurities using a purification method such as recrystallization as required.
• the tripeptide compound of the present invention is prepared from the intermediate material represented by the general formula (XIII), an amino acid represented by the general formula (XXII), a carboxylic acid represented by the general formula (XXIV), and the like as raw materials, the molecular structure can be easily identified by means of a spectroscopy technique such as a nuclear magnetic resonance method or an infrared absorption method with reference to each configuration respectively originated from these compounds.
• the tripeptide compound of the present invention which is represented by the general formula (III) is the same as the compound represented by the above general formula (VII) except that the C-terminal substituted benzyl group is replaced by a tert-butyl group, the synthesis itself can be carried out by a completely similar process. Specifically, almost the same synthetic conditions can be employed except for the use of a tert-butylamine instead of a substituted benzylamine which is one of the starting materials so that the synthesis can be easily carried out.
• the tripeptide compound of the present invention can be administered as a medicine using a commonly used carrier for medicine or an excipient according to a conventional method when clinically applied as an anti-AIDS medicine.
• the tripeptide compound of the present invention can be used in the form of, for example, an injection agent such as a muscle injection agent or an intravenous injection agent, a parenteral administration agent such as a spray agent or a suppository, or an oral administration agent such as a granulars, capsules, or tablets.
• the medicine is administered at a dose in the range of usually 0.5-50 mg/kg, and preferably 3-30 mg/kg, for an adult 2 to 4 times a day, although a specific dose is appropriately determined according to the symptom of the subject, the purpose of treatment such as controlling AIDS symtoms from exhibiting or progressing, the age and sex of the subject.
• a dosage form applied to oral administration of various synthetic peptide compounds which have already been proposed as the HIV protease inhibitor can be employed (see, for example, Japanese Patent Application Laid-open No. 208478/1996).
• the dipeptide compound of the present invention and the process for preparing the compound will now be described in detail by way of examples. Also described are examples showing the characteristics of the tripeptide compound of the present invention such as superior anti-HIV activity due to the high HIV protease inhibition action and low cytotoxic properties which make this compound suitable as a medicine. These examples should by no means be construed as limiting the present invention.
• Apns ((2S,3S)-3-amino-2-hydroxy-4-phenylbutanoic acid; (2S,3S)-H-AHPBA), cyclic ⁇ -amino acids such as Thz ((R)-1,3-thiazolidine-4-carboxylic acid), Dmt ((R)-5,5-dimethyl-1,3-thiazolidine-4-carboxylic acid), Mox ((4S,5R)-5-methyl-1,3-oxazolidine-4-carboxylic acid), Pip ((R)-pipecolic acid; (R)-2-piperidine carboxylic acid), and the like, as well as other ⁇ -amino acids, used as raw materials in the examples were previously prepared as amino acid N-protected derivatives of these compounds according to the method known in the art by publication, followed by release of the amino group.
• Thz ((R)-1,3-thiazolidine-4-carboxylic acid), Dmt ((R)-5,5
• acyl chlorides were used for Z group (benzyloxycarbonyl group), methoxycarbonyl group, and ethoxycarbonyl group; acid anhydrides were used for acetyl group; corresponding carboxylic acids or derivatives thereof were used for acyl group such as methoxyacetyl group, ethoxyacetyl group, iQoa group (5-isoquinolyloxyacetyl group; 5-isoquinolyloxyethanoyl group), 2-chromonecarbonyl group (benzo-4-pyron-2-ylcarbonyl group; Chc), benzofuran-2-ylcarbonyl group (Bfc) and the like.
• some commercially available intermediate raw material compounds such as amino acid derivatives of which the nitrogen was protected and modified by a Z group, were used.
• EDC ⁇ HCl (6.30 g, 33.0 mmol) was added to a solution of Boc-Thz-OH (6.99 g, 30 mmol), 2-methylbenzylamine (4.46 ml, 36.0 mmol), and HOBt (4.05 g, 30 mmol) in CH 2 Cl 2 (100 ml) while cooling with ice, and the mixture was stirred overnight.
• the reaction mixture was washed with 3% Na 2 CO 3 (twice), 1N HCl (once), and 5% NaCl (once) and dried over MgSO 4 . After filtration and concentration, the residue was dissolved in CH 2 Cl 2 (100 ml).
• the title compound was synthesized from H-Dmt-NHBzl(2-Cl) (0.14g) obtained in the step 1, Z-Asn-Apns-OH (0.22 g), HOBt (68 mg), and EDC ⁇ HCl (105 mg) according to the step 2 of Example 1 (287 mg).
• HOBt (0.41 g), Boc-Asn-ONp (1.27 g), and Et 3 N (0.92 ml) were added to a solution of H-Apns-Dmt-NHBzl(2-Me) (1.32 g) obtained in the step 2 of Example 3 in DMF (20 ml) and the mixture was stirred overnight. After the addition of ethyl acetate, the reaction mixture was washed with 3% NaHCO 3 , 1N HCl, and 5% NaCl and dried over MgSO 4 . After filtration and concentration, the residue was recrystallized from AcOEt/n-hexane to obtain the title compound (1.79 g).
• Boc-Val-OH (0.68 g), HOBt (0.43 g), and EDC ⁇ HCl (0.60 g) were added to a solution of H-Apns-Dmt-NHBzl(2-Me) (1.32 g) obtained in the step 2 of Example 3 in DMF (10 ml) and the mixture was stirred overnight. After the addition of ethyl acetate, the reaction mixture was washed with 3% NaHCO 3 , 1N HCl, and 5% NaCl and dried over MgSO 4 . After filtration and concentration, the residue was recrystallized from ethyl acetate/n-hexane to obtain the title compound (1.82 g).
• the compound Boc-Val-Apns-Dmt-NHtBu (148 mg) obtained in the step 1 was treated with 4N HCl/dioxane for two hours at room temperature. After concentration, the mixture was dissoleved in DMF and neutralized with Et 3 N (37 ml). 3-dimethylaminophenoxyacetic acid (51 mg), HOBt (35 mg), and EDC ⁇ HCl (53 mg) were added to a solution of the resulting H-Val-Apns-Dmt-NHtBu in DMF and the mixture was stirred overnight.
• Methanesulfonyl chloride (25 ⁇ l) and triethylamine (46 ⁇ l) were added to a solution of H-Val-Apns-Dmt-NHBzl(2-Me) (162 mg) in methylene chloride (3 ml) and the mixture was stirred for two hours. After the addition of ethyl acetate, the reaction mixture was washed with 3% Na 2 CO 3 , 1N HCl, and 5% NaCl and dried over MgSO 4 . After filtration and concentration, the residue was purified using silica gel column chromatography and reprecipitated from ethyl acetate/n-hexane to obtain the title compound (127 mg).
• Methanesulfonyl chroride (25 ⁇ l) and triethylamine (46 ⁇ l) were added to a solution of H-Abu-Apns-Dmt-NHBzl(2-Me) (158 mg) in ethyl acetate (3 ml) and the mixture was stirred for two hours. After the addition of ethyl acetate, the reaction mixture was washed with 3% Na 2 CO 3 , 1N HCl, and 5% NaCl and dried over MgSO 4 . After filtration and concentration, the residue was purified using silica gel column chromatography and reprecipitated from ethyl acetate/n-hexane to obtain the title compound (92 mg).
• the tripeptide compound of the present invention has superior HIV protease inhibition activity and, in particular, is suitable for use as a medication because of its remarkably low inhibition constant to the HIV protease, the following experiments were performed.
• the compounds of the Examples 1-38 were evaluated according to previously published experimental methods (see, for example, Journal of Synthetic Organic Chemistry Japan vol. 52, 403-412 (1994) and Japanese Patent Application Laid-open No. 170722/1993).
• the protease activity was measured using a recombinant HIV-1 protease (see Biochemistry, 250 (9)-264 (1990)) and a synthesized peptide substrate.
• the inhibition rate was calculated by determining a peptide fragment generated by cleavage between Tyr-Pro of the synthesized substrate after the reaction of 60 min at 37°C using a reversed phase HPLC in the presence of the test compounds in various concentrations (see, for example, Japanese Patent Application Laid-open No. 170722/1993).
• Table 1 Examples of the evaluation results of the HIV protease inhibition activity of the tripeptide compound of the present invention by the above-described method is shown in Table 1. As shown in these results, each tripeptide compound of the present invention exhibits significantly high HIV protease inhibition activity.
• the concentration of the test compounds in Table 1 represents the final concentration in the reaction mixture.
• Example 1 Z-Asn-Apns-Dmt-NHBzl 96.0
• Example 2 Z-Asn-Apns-Thz-NHBzl(2-Me) 71.7
• Example 3 Z-Asn-Apns-Dmt-NHBzl(2-Me) 100.0
• Example 4 Z-Val-Apns-Dmt-NHBzl(2-Me) 95.3
• Example 5 Z-Asn-Apns-Dmt-NHBzl(2-Cl) 98.4
• Example 6 Chc-Asn-Apns-Dmt-NHBzl(2-Me) 98.4
• Example 7 Bfc-Asn-Apns-Dmt-NHBzl(2-Me) 91.2
• Example 8 iQoa-Val-Apns-Dmt-NHBzl(2-Me) 97.
• the enzyme inhibition constant Ki was evaluated in order to verify that the tripeptide compound of the present invention exhibits a markedly superior inhibition ability compared with a conventional tripeptide type inhibition substance.
• the known compound KNI-162 for which high HIV protease inhibition activity has been reported to publications see, for example, Yoshiaki Kiso, Journal of Synthetic Organic Chemistry Japan vol. 52, 403-412 (1994) was similarly tested for comparison.
• This compound KNI-162 (R) 3 ⁇ (2S,3S)-3-[N-(benzyloxycarbonyl)-L-asparaginyl]amino-2-hydroxy-4-phenylbutanoyl ⁇ -5,5-dimethyl-1,3-thiazolidine-4-carboxamide is a hydroxymethylcarboxamide type tripeptide compound having a (2S,3S)-3-amino-2-hydroxy-4-phenylbutanoyl skeleton which is similar to the tripeptide compound of the present invention except that the tert-butyl group is substituted on the C-terminal amide nitrogen atom.
• the synthesized peptide substrate having a sequence of H-Lys-Ala-Arg-Val-Tyr-Phe(4-NO 2 )-Glu-Ala-Nle-NH 2 was used in this experiment.
• the enzyme reaction was measured using a reversed phase HPLC by determining the C-terminal peptide fraction produced by cleavage just behind Tyr in the synthesized substrate which is obtained by reacting the following composition for 15 min. at 37°C and stopping the reaction by the addition of 1N HCl (75 ⁇ l).
• Recombinant HIV-1 protease enzyme solution 10 ⁇ l 0.5 mM synthetic substrate aqueous solution 10 ⁇ l DMSO Inhibitor solution 5 ⁇ l 0.2 M MES solution (1 M NaCl, 2 mM EDTA-2Na, 2 mM DTT, pH 5.5) 25 ⁇ l
• the enzyme inhibition constant Ki of the tripeptide compound of the present invention exhibits remarkably low values. Specifically, it is recognized that the tripeptide compound of the present invention has a remarkably high binding affinity with the HIV protease. Moreover, compared with the compound KNI-162 of the Reference Example, it is judged that the tripeptide compound of the present invention exhibits remarkably superior HIV protease inhibition activity. It is considered that the increase of the binding affinity of the tripeptide compound of the present invention is mainly caused by the C-terminal substituted benzyl amide.
• the tripeptide compound of the present invention excels in the effect of inhibiting production of the HIV virus particles in the host cell owing to the remarkably superior HIV protease inhibition compared with the conventional tripeptide type inhibition substance.
• the following anti-HIV activity evaluation tests were carried out.
• 50% cytotoxic concentration was evaluated in order to identify the degree of cytotoxicity.
• Ritonavir which is sold at a market peptide type HIV protease inhibition substance, was also evaluated for comparison.
• the anti-HIV activity was evaluated using a CEM-SS cell as the host cell and the HIV virus strain HIV-1 IIIB (see, for example, the European Patent Publication No. EP 0751145 A2).
• test compounds were added to the culture medium in various concentrations, then the HIV infected CEM-SS cell was added and incubated for six days at 37°C using a CO 2 incubator. The number of the living cells was then measured by a XTT method.
• the test compounds were added in various concentrations and the HIV virus non-infected cell was incubated under similar conditions.
• the HIV infected CEM-SS cell was incubated without the addition of the test compounds.
• the HIV virus non-infected cell was incubated without the addition of the test compound as the control group.
• the anti-virus activity was evaluated in a concentration in which the cytotoxic effect caused by the HIV infected virus in the above HIV infected cell group was inhibited 50 percent (EC 50 , 50% effective concentration), and the cytotoxicity was evaluated in a concentration in which the cytotoxic effect by the addition of the test compound in the HIV virus non-infected cell group occurred at 50 percent (TC 50 , 50% cytotoxic concentration) respectively.
• TC 50 50% cytotoxic concentration
• the tripeptide compound of the present invention exhibits the anti-virus activity in markedly low concentrations corresponding to the significantly small values of the enzyme inhibition constant Ki.
• the cytotoxicity is only shown in concentrations three digits or more in the range of concentration in which the anti-virus activity is exhibited.
• the metabolic characteristics of the tripeptide compound of the present invention were evaluated using rats for a test animal.
• the result of KNI-272 ((R)-N-tert-butyl-3-((2S,3S)-2-hydroxy-3-[N-(isoquinolin-5-yloxy)acetyl-methylthio-L-alanyl]amino-4-phenylbutanoyl ⁇ -1,3-thiazolidine-4-carboxamide; iQoa-Mta-Apns-Thz-NHtBu) which is a tripeptide derivative known in the art, is also shown.
• the amount administred was 10 mg/kg.
• the evaluated administration methods are a method (id) of administering a solution of a fixed amount of the test compound in the duodenum of a test animal, and a method (iv) of administering the solution intravenously.
• blood samples were taken from the test animal and concentrations of the residual test compound in the plasma were analyzed.
• AUC area under medicine concentration curve in plasma
• MRT mean residence time
• t 1/2 ⁇
• ⁇ final phase half-life period
• each tripeptide compound of the present invention has superior stability in vivo and is able to maintain high concentration in plasma compared with KNI-272 which is the compound of the Control Example.
• a granular agent was prepared according to the method of Japanese Patent Application Laid-open No. 208478/1996 using the tripeptide compound of the present invention as the effective component.
• One of the examples is given below.
• Chc-Asn-Apns-Dmt-NHBzl(2-Me) 50 g obtained in Example 6 and hydroxypropyl methylcellulose 2910 (binder) (20 g) were dissolved in a mixture (930 g) of ethanol (The Pharmacopoeia of Japan) (837 g) and purified water (93 g) to adjust the total weight to 1000 g.
• the resulting solution was used as a coating solution.
• True spherical particles with a diameter of 150-300 ⁇ m, Cellphia CP203 (500 g) (trademark: manufactured by Asahi Chemical Industry Co., Ltd.), was used as a core material.
• the particles were fed to a rolling-type fluidized bed coating apparatus Multiplex (trademark: manufactured by Paureck Co., Ltd.) under the conditions of air feed temperature of 55°C and air flow rate of 40 m 3 /hr.
• the above coating solution was sprayed and dried to form a coating layer on the core material particles.
• These coating particles were sieved using a 42-mesh sieve and the particles which passed through were used as the granular agent.
• the tripeptide compound of the present invention which is represented by the general formula (I) exhibits peculiar and remarkably high HIV protease inhibition activity, because not only the dipeptide moiety part which is indispensable for the HIV protease inhibition activity, but also the substituted benzyl group on the C-terminal amide nitrogen atom participate in binding to the enzyme active center of the HIV protease. Therefore, when used for clinical application as an anti-AIDS medicine, there is the advantage that the amount to be administered to obtain the desired concentration of the peptide compound, which is the effective component for producing the desired medical effect, can be decreased.
• the tripeptide compound which is represented by the general formula (III) exhibits peculiar and remarkably high HIV protease inhibition activity as well as high digestive tract absorption, when used for a clinical application as the anti-AIDS medicine, there is the advantage that the amount to be administered to obtain the desired concentration of the peptide compound, which is the effective component for producing the desired medical effect, can be decreased.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Crystallography & Structural Chemistry (AREA)
• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Biochemistry (AREA)
• Biophysics (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Medicinal Chemistry (AREA)
• Molecular Biology (AREA)
• Proteomics, Peptides & Aminoacids (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Peptides Or Proteins (AREA)

Applications Claiming Priority (5)

Publications (2)

Family

ID=26480090

Family Applications (1)

Country Status (5)

Cited By (4)

Families Citing this family (2)

Citations (2)

Family Cites Families (37)

• 1997
  • 1997-12-22 EP EP97949191A patent/EP0900566A4/de not_active Withdrawn
  • 1997-12-22 WO PCT/JP1997/004734 patent/WO1998029118A1/ja not_active Application Discontinuation
  • 1997-12-22 US US09/155,773 patent/US6291432B1/en not_active Expired - Fee Related
  • 1997-12-22 CA CA002249747A patent/CA2249747A1/en not_active Abandoned
• 1998
  • 1998-09-16 NO NO984284A patent/NO984284L/no not_active Application Discontinuation

Patent Citations (2)

Non-Patent Citations (2)

Also Published As

Similar Documents

Legal Events